Specific initiation of transcription of AMV and MC29 cloned DNAs by RNA polymerase II (alpha-Amanitin sensitive) in a eucaryotic cell-free system has been obtained. The site of initiation of transcription is located within the U3 region of the LTR sequences of both AMV and MC29. These results indicate that the information necessary for retroviral transcription is located within the viral genomes. In addition, initiation of transcription from a corresponding canonical Goldberg-Hogness sequence in the myb gene of AMV has been obtained, suggesting that this conserved sequence element may serve as an independent promoter. Current studies are designed to elucidate the role of 5'-extragenic regions in the expression of retroviruses. Deletion mutants harboring mutated upstream sequences are being constructed using the Bal-3l enzyme system and recombinant plasmid technology.